Silane mixtures

ABSTRACT

Silanation compositions containing a mixture of two or more silanation reagents, where at least one silanation reagent includes a functional group capable of supporting polymer synthesis and at least one silanation reagent includes no functional group capable of supporting polymer synthesis are useful in modulating the active site density and hydrolytic stability of a surface. These compositions are particularly useful in silanating a surface prior to preparation of a polymer array and provide for increased hybridization results.

RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.12/358,472 filed Jan. 23, 2009, which is a continuation of U.S.application Ser. No. 11/442,680 filed May 25, 2006, now abandoned. Eachapplication is hereby incorporated by reference herein in its entiretyfor all purposes.

Application Ser. No. 12/014,879 filed Jan. 16, 2008, now U.S. Pat. No.7,790,389 and U.S. application Ser. No. 10/177,169 filed Jun. 20, 2002,now U.S. Pat. No. 7,332,273 are incorporated by reference in theirentirety for all purposes.

BACKGROUND OF THE INVENTION

Silanating reagents have been developed which react with and coatsurfaces, such as silica surfaces. Monofunctional silanating reagentshave been used to form monolayer surface coatings, while di- andtri-functional silanating reagents have been used to form polymerizedcoatings on silica surfaces, providing reaction sites for covalentattachment of various materials to the surface. Many silanatingreagents, however, produce coatings with undesirable propertiesincluding instability to hydrolysis and an inadequate ability to maskthe silica surface.

SUMMARY OF THE INVENTION

The present invention provides silanation compositions that include amixture of two or more silanation reagents, where at least onesilanation reagent includes a functional group capable of supportingpolymer synthesis and at least one silanation reagent includes nofunctional group capable of supporting polymer synthesis.

Silanation reagent compositions (mixtures), one reagent having afunctional group capable of supporting polymer synthesis and anothersilanation reagent having no functional group capable of supportingpolymer synthesis, allows for control of the density of polymers on asubstrate. An overly high density of polymers on a substrate, e.g., anarray of oligonucleotides (probes) on a glass chip, where the array isto be used for determining binding of complementary nucleic acids or“targets,” has undesirable properties. Target binding is inhibited by anoverly dense probe concentration on the surface of the substrate bysteric and electrostatic repulsive forces of overly crowded probes onthe surface of the substrate. Where the targets are labeled withfluorescent molecules, for example, inhibition of target binding in turnleads to an undesirable decrease in hybridization signal, which in turncould result in false negatives.

It has been discovered in accordance with the present invention that bysilanating with a mixture of functional and non-functional silanatingreagents a silane coating is created having increased hydrolyticstability. Merely reducing the density of functional silanes withoutadding non-functional silanes would result in silane coatings withreduced hydrolytic stability.

In one preferred embodiment, the silanation composition has at least onesilanation reagent which includes a functional group capable ofsupporting polymer synthesis is selected from the group consisting of:

and wherein the at least one silane reagent which does not include afunctional group capable of supporting polymer synthesis is selectedfrom the group consisting of alkyltrimethoxysilane,1,2-bis(trimethoxysilyl)ethane, 1,3-bis(trimethoxysilyl)propane,1,6-bis(trimethoxysilyl)hexane,3-(N,N-dimethylamino)propyltrimethoxysilane andN,N-bis(3-trimethoxysilylpropyl)methylamine.

In one preferred embodiment, the silanation reagent including afunctional group capable of supporting polymer synthesis is a maskedsilanation reagent. Many silanes with masked hydroxyl groups, forexample, are highly volatile. Volatile, masked silanes can be readilypurified by, e.g., distillation, and can be readily employed ingas-phase deposition methods of silanating substrate surfaces. Gas-phasedeposition, as opposed to dipping methods provide for more efficientdeposition and uniform coating of the substrate. Prior to attachingmonomers the masked silanating reagent must be demasked by proceduresdiscussed, infra.

The present invention provides silanation compositions that include amixture of two or more silanation reagents, where at least onesilanation reagent includes a functional group capable of supportingpolymer synthesis and at least one silanation reagent includes nofunctional group capable of supporting polymer synthesis inpredetermined ratios. In a preferred embodiment, the ratios are weightto volume ratios (W:V). In another preferred embodiment, the rations aremolar ratios.

The present invention provides methods of silanating a surface thatcomprises contacting a silica or other silicon oxide-containing surfacewith a silanation composition that includes a mixture of two or moresilanation reagent, where at least one silanation reagent includes afunctional group capable of supporting polymer synthesis and at leastone silanation reagent includes no functional group capable ofsupporting polymer synthesis.

In a further aspect, the present invention provides a method ofpreparing an array of polymers that include the steps of providing asubstrate, contacting a silica or other silicon oxide-containing surfaceof the substrate with a silanation composition that includes a mixtureof two or more silanation reagents, where at least one silanationreagent includes a functional group capable of supporting polymersynthesis and at least one silanation reagent includes no functionalgroup capable of supporting polymer synthesis; protecting the functionalgroup capable of supporting polymer synthesis with a protecting group orreacting the functional group capable of supporting polymer synthesiswith a compound having a reactive group protected by a protecting group;removing the protecting group in selected regions of the surface toprovide an exposed functional or reactive group; reacting said exposedgroup with a monomer, wherein the monomer is coupled to the exposedgroup; and repeating the steps of removing and reacting to produce thearray of polymers.

The present invention also provides for an array of polymers on asurface of a substrate, where the surface of the substrate is silanatedby a silanation composition that includes a mixture of two or moresilanation reagents, where at least one silanation reagent includes afunctional group capable of supporting polymer synthesis and at leastone silanation reagent includes no functional group capable ofsupporting polymer synthesis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Depicts two non functional silane compounds(Bis(3-methylamino)propyl)trimethoxysilane (R2) andBis(trimethoxysilyl)ethane (BTMSE)) and two functional silanes(N-(hydroxyethyl)N-methylamino propyltrimethoxysilane (R2) andN-Hydroxyethyl-N,N-Bis(Trimethoxysilylpropyl)amine).

FIG. 2 Depicts formulations for Silanation of various mixtures ofR2+Bisb.

FIG. 3 Depicts functional hydroxyl density determination for variousmixtures of R2 and Bisb.

FIG. 4 depicts formulations for silanation of R1 and R2.

FIG. 5 depicts functional hydroxyl density determinations for variousR2/R1 mixtures.

FIG. 6 depicts high-efficiency PAG/DMT synthesis of 3 μm array in a25mer test pattern and the corresponding hybridization signals.

DETAILED DESCRIPTION OF THE INVENTION

The present invention has many preferred embodiments and relies on manypatents, applications and other references for details known to those ofthe art. Therefore, when a patent, application, or other reference iscited or repeated below, it should be understood that it is incorporatedby reference in its entirety for all purposes as well as for theproposition that is recited.

As used in this application, the singular form “a,” “an,” and “the”include plural references unless the context clearly dictates otherwise.For example, the term “a reagent” includes a plurality of reagents,including mixtures thereof.

An individual is not limited to a human being but may also be otherorganisms including but not limited to mammals, plants, bacteria, orcells derived from any of the above.

Throughout this disclosure, various aspects of this invention can bepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

The practice of the present invention may employ, unless otherwiseindicated, conventional techniques of organic chemistry, polymertechnology, molecular biology (including recombinant nucleic acidtechniques), cell biology, biochemistry, and immunology as would beunderstood by one of the ordinary skill. Such conventional techniquesinclude polymer array synthesis, hybridization, ligation, and detectionof hybridization using a label. Specific illustrations of suitabletechniques can be had by reference to the examples herein below.However, other equivalent conventional procedures can, of course, alsobe used. Such conventional techniques and descriptions can be found instandard laboratory manuals such as Genome Analysis: A Laboratory ManualSeries (Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells: ALaboratory Manual, PCR Primer: A Laboratory Manual, and MolecularCloning: A Laboratory Manual (all from Cold Spring Harbor LaboratoryPress), Stryer, L. (1995) Biochemistry (4th Ed.) Freeman, New York,Gait, “Oligonucleotide Synthesis: A Practical Approach” 1984, IRL Press,London, Nelson and Cox (2000), Lehninger, Principles of Biochemistry3^(rd) Ed., W.H. Freeman Pub., New York, N.Y. and Berg et al. (2002)Biochemistry, 5^(th) Ed., W.H. Freeman Pub., New York, N.Y., all ofwhich are herein incorporated by reference in their entirety.

The present invention can employ solid substrates, including arrays insome preferred embodiments. Methods and techniques applicable to polymer(including protein) array synthesis have been described in U.S. Ser. No.09/536,841, WO 00/58516, U.S. Pat. Nos. 5,143,854, 5,242,974, 5,252,743,5,324,633, 5,384,261, 5,405,783, 5,424,186, 5,451,683, 5,482,867,5,491,074, 5,527,681, 5,550,215, 5,571,639, 5,578,832, 5,593,839,5,599,695, 5,624,711, 5,631,734, 5,795,716, 5,831,070, 5,837,832,5,856,101, 5,858,659, 5,936,324, 5,968,740, 5,974,164, 5,981,185,5,981,956, 6,025,601, 6,033,860, 6,040,193, 6,090,555, 6,136,269,6,269,846 and 6,428,752, in PCT Applications Nos. PCT/US99/00730(International Publication Number WO 99/36760) and PCT/US01/04285(International Publication Number WO 01/58593), which are allincorporated herein by reference in their entirety.

A “substrate” is a material having a rigid, semi-rigid or gelatinoussurface. Typical examples include glass or suitable polymer materials.In some embodiments of the present invention, at least one surface ofthe substrate will be substantially flat, although in some embodimentsit may be desirable to physically separate synthesis regions fordifferent polymers with, for example, wells, raised regions, etchedtrenches, or the like. In some embodiments, the substrate itselfcontains wells, trenches, flow through regions, etc. which form all orpart of the synthesis regions. According to other embodiments, smallbeads may be provided on the surface, and compounds synthesized thereonoptionally may be released upon completion of the synthesis. Substratesare well known in the art and are readily commercially available throughvendors such as USPG, PPG Industries, AFG Industries and others. Thesubstrates used in the invention are preferably those that are readilysilanated, such as glass, fused silica and silicon wafers.

Patents that describe synthesis techniques in specific embodimentsinclude U.S. Pat. Nos. 5,412,087, 6,147,205, 6,262,216, 6,310,189,5,889,165, and 5,959,098, which are all incorporated by reference intheir entirety. Nucleic acid arrays are described in many of the abovepatents, but the same general methodologies are applicable topolypeptide arrays.

The present invention also contemplates many uses for polymers attachedto substrates. These uses include gene expression monitoring, profiling,library screening, genotyping and diagnostics. Gene expressionmonitoring, and profiling methods can be shown in U.S. Pat. Nos.5,800,992, 6,013,449, 6,020,135, 6,033,860, 6,040,138, 6,177,248 and6,309,822, which are all incorporated by reference in their entirety.Genotyping and uses therefore are shown in U.S. Ser. Nos. 60/319,253,10/013,598 (U.S. Patent Application Publication 20030036069), and U.S.Pat. Nos. 5,856,092, 6,300,063, 5,858,659, 6,284,460, 6,361,947,6,368,799 and 6,333,179, which are incorporated by reference in theirentirety. Other uses are embodied in U.S. Pat. Nos. 5,871,928,5,902,723, 6,045,996, 5,541,061, and 6,197,506, which are incorporatedby reference in their entirety.

The present invention also contemplates sample preparation methods incertain preferred embodiments. Prior to or concurrent with genotyping,the genomic sample may be amplified by a variety of mechanisms, some ofwhich may employ PCR. See, e.g., PCR Technology: Principles andApplications for DNA Amplification (Ed. H. A. Erlich, Freeman Press, NY,N.Y., 1992); PCR Protocols: A Guide to Methods and Applications (Eds.Innis, et al., Academic Press, San Diego, Calif., 1990); Mattila et al.,Nucleic Acids Res. 19, 4967 (1991); Eckert et al., PCR Methods andApplications 1, 17 (1991); PCR (Eds. McPherson et al., IRL Press,Oxford); and U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159 4,965,188,and 5,333,675, and each of which is incorporated herein by reference intheir entirety. The sample may be amplified on the array. See, forexample, U.S. Pat. No. 6,300,070 and U.S. Ser. No. 09/513,300, which areincorporated herein by reference in their entirety.

Other suitable amplification methods include the ligase chain reaction(LCR) (e.g., Wu and Wallace, Genomics 4, 560 (1989), Landegren et al.,Science 241, 1077 (1988) and Barringer et al. Gene 89:117 (1990)),transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA 86,1173 (1989) and WO 88/10315), self-sustained sequence replication(Guatelli et al., Proc. Nat. Acad. Sci. USA, 87, 1874 (1990) and WO90/06995), selective amplification of target polynucleotide sequences(U.S. Pat. No. 6,410,276), consensus sequence primed polymerase chainreaction (CP-PCR) (U.S. Pat. No. 4,437,975), arbitrarily primedpolymerase chain reaction (AP-PCR) (U.S. Pat. Nos. 5,413,909, 5,861,245)and nucleic acid based sequence amplification (NABSA). (See, U.S. Pat.Nos. 5,409,818, 5,554,517, and 6,063,603, each of which is incorporatedherein by reference). Other amplification methods that may be used aredescribed in, U.S. Pat. Nos. 5,242,794, 5,494,810, 4,988,617 and in U.S.Ser. No. 09/854,317. Each of the above references is incorporated hereinby reference in its entirety.

Additional methods of sample preparation and techniques for reducing thecomplexity of a nucleic sample are described in Dong et al., GenomeResearch 11, 1418 (2001), in U.S. Pat. Nos. 6,361,947, 6,391,592 andU.S. Ser. Nos. 09/916,135, 09/920,491 (U.S. Patent ApplicationPublication 20030096235), Ser. No. 09/910,292 (U.S. Patent ApplicationPublication 20030082543), and Ser. No. 10/013,598, each of which isincorporated herein by reference in its entirety.

Numerous methods for conducting polynucleotide hybridization assays havebeen well developed. Hybridization assay procedures and conditions willvary depending on the application and are selected in accordance withthe general binding methods known including those referred to in:Maniatis et al. Molecular Cloning: A Laboratory Manual (2^(nd) Ed. ColdSpring Harbor, N.Y., 1989); Berger and Kimmel Methods in Enzymology,Vol. 152, Guide to Molecular Cloning Techniques (Academic Press, Inc.,San Diego, Calif., 1987); Young and Davism, P.N.A.S, 80: 1194 (1983).Methods and apparatus for carrying out repeated and controlledhybridization reactions have been described in U.S. Pat. Nos. 5,871,928,5,874,219, 6,045,996 and 6,386,749, 6,391,623 each of which is herebyincorporated by reference in its entirety.

The present invention contemplates detection of hybridization between aligand and its corresponding receptor by generation of specific signals.See U.S. Pat. Nos. 5,143,854, 5,578,832; 5,631,734; 5,834,758;5,936,324; 5,981,956; 6,025,601; 6,141,096; 6,185,030; 6,201,639;6,218,803; and 6,225,625, in U.S. Ser. No. 60/364,731 and in PCTApplication PCT/US99/06097 (published as WO99/47964), each of which alsois hereby incorporated by reference in its entirety. Each of thesereferences is incorporated herein by reference in its entirety.

Methods and apparatus for signal detection and processing of intensitydata are disclosed in, for example, U.S. Pat. Nos. 5,143,854, 5,547,839,5,578,832, 5,631,734, 5,800,992, 5,834,758; 5,856,092, 5,902,723,5,936,324, 5,981,956, 6,025,601, 6,090,555, 6,141,096, 6,185,030,6,201,639; 6,218,803; and 6,225,625, in U.S. Ser. No. 60/364,731 and inPCT Application PCT/US99/06097 (published as WO 99/47964), each of whichalso is hereby incorporated by reference in its entirety.

The practice of the present invention may also employ conventionalbiology methods, software and systems. Computer software products of theinvention typically include computer readable medium havingcomputer-executable instructions for performing the logic steps of themethod of the invention. Suitable computer readable medium includefloppy disk, CD-ROM/DVD/DVD-ROM, hard-disk drive, flash memory, ROM/RAM,magnetic tapes and etc. The computer executable instructions may bewritten in a suitable computer language or combination of severallanguages. Basic computational biology methods are described in, e.g.Setubal and Meidanis et al., Introduction to Computational BiologyMethods (PWS Publishing Company, Boston, 1997); Salzberg, Searles,Kasif, (Ed.), Computational Methods in Molecular Biology, (Elsevier,Amsterdam, 1998); Rashidi and Buehler, Bioinformatics Basics:Application in Biological Science and Medicine (CRC Press, London, 2000)and Ouelette and Bzevanis Bioinformatics: A Practical Guide for Analysisof Gene and Proteins (Wiley & Sons, Inc., 2^(nd) ed., 2001). See U.S.Pat. No. 6,420,108. Each of these references is incorporated herein byreference in its entirety.

The present invention may also make use of various computer programproducts and software for a variety of purposes, such as probe design,management of data, analysis, and instrument operation. See, U.S. Pat.Nos. 5,593,839, 5,795,716, 5,733,729, 5,974,164, 6,066,454, 6,090,555,6,185,561, 6,188,783, 6,223,127, 6,229,911 and 6,308,170. Each of thesereferences is incorporated herein by reference in its entirety.

Light patterns can also be generated using Digital Micromirrors, LightCrystal on Silicon (LCOS), light valve arrays, laser beam patterns andother devices suitable for direct-write photolithography. See. e.g.,U.S. Pat. Nos. 6,271,957 and 6,480,324, incorporated herein byreference.

Additionally, the present invention may have preferred embodiments thatinclude methods for providing genetic information over networks such asthe Internet as shown in U.S. Ser. No. 10/063,559 (United StatesPublication No. 20020183936), U.S. Provisional Applications 60/349,546,60/376,003, 60/394,574 and 60/403,381). Each of these references isincorporated herein by reference in its entirety.

The present invention provides methods, devices, and compositions forthe formation of arrays of large numbers of different polymer sequences.In one aspect of the present invention, the methods and compositionsprovided herein involve the conversion of radiation signals intochemical products that are particularly useful in polymer synthesis. Theinvention also includes the arrays formed using the methods andcompositions disclosed herein. One aspect of the invention includesmethods, compositions, and devices for the synthesis of an array ofdifferent polymers in selected and predefined regions of a substrate.Another aspect of the invention includes those arrays and variousmethods of using them.

Such arrays are used in, for example, in nucleic acid analysis.Polynucleotide or nucleic acid arrays are especially suitable forchecking the accuracy of previously elucidated sequences and fordetecting mutations and polymorphisms. Polymer arrays are also used inscreening studies to evaluate their interaction with, for example,receptors such as antibodies in the case of peptide arrays or withnucleic acids in the case, for example of oligonucleotide arrays. Forexample, certain embodiments of the invention provide for the screeningof peptides to determine which if any of a diverse set of peptides hasstrong binding affinity with a receptor.

In some embodiments of the present invention, the arrays formed by thepresent invention are used in competitive assays or other well-knowntechniques to screen for compounds having certain activities. Forexample, vast collections of synthetic or natural compounds areimmobilized on predefined regions of a substrate. The reaction of theimmobilized compounds (or compound) with various test compositions suchas the members of a chemical library or a biological extract are testedby dispensing small aliquots of each member of the library or extract toa different region. In one embodiment, a large collection of humanreceptors is deposited on a substrate, one in each region to form anarray. A plant or animal extract is then screened for binding to variousreceptors of the array.

Nucleic acid sequences can also be immobilized in specific locations orpredefined regions of a substrate using the current invention. In someembodiments, such immobilized nucleic acid arrays are used inhybridization assays for gene expression monitoring, nucleic acidamplifications, nucleic acid computation, and nucleic acid analysis ingeneral.

A “predefined region” is a localized area on a substrate which is, was,or is intended to be used for formation of a selected polymer and isotherwise referred to herein in the alternative as “reaction” region, a“selected” region, simply a “region” or a “feature”. The predefinedregion may have any convenient shape, e.g., circular, rectangular,elliptical, wedge-shaped, etc. In accordance with the present invention,the arrays of the present invention have features on the order of 10-100μm, i.e. 10×10 μm² to 100×100 μm² for approximately square features.More preferably the features will be on the order of 1-10 μm. It is alsoan object of the present invention to provide features having sub-microndimensions. Such features are preferably on the order of 100-1000 nm.Within these regions, the polymer synthesized therein is preferablysynthesized in a substantially pure form. However, in other embodimentsof the invention, predefined regions may substantially overlap. In suchembodiments, hybridization results may be resolved by software forexample.

The density of active sites (i.e., sites available for derivatization)within a feature is preferably at least 5 pmol/cm², but is morepreferably at least 10 pmol/cm² or even 20 pmol/cm². Typically, thedensity of active sites is from 5-20 pmol/cm².

The present invention has certain features in common with the radiationdirected methods discussed in U.S. Pat. No. 5,143,854, incorporatedherein by reference. The radiation-directed methods discussed in thatpatent involve activating predefined regions of the substrate and thencontacting the substrate with a preselected monomer solution. Thepredefined regions can be activated with, for example, a light sourceshown through a mask (much in the manner of photolithographic techniquesused in integrated circuit fabrication). Other regions of the substrateremain inactive because they are blocked by the mask from illumination.Thus, a light pattern defines which regions of the substrate react witha given monomer. By repeatedly activating different sets of predefinedregions and providing different monomer compositions thereto, a diversearray of polymers is produced on or near the substrate.

According to another aspect of the present invention, there is norequirement for the use of masks. Predefined regions of the array may beactivated by light without the use of photomasks, for example withoutlimitation, by spatial light modulation as discussed in U.S. Pat. No.6,271,957 and related applications (parent and progeny patents).

An “alkoxy” groups refers to an alkane linked to an oxygen, R—O—, whereR is an alkyl group. In accordance with an aspect of the presentinvention, when an alkoxy group is attached to a silane, it has thestructure —Si—O—R, wherein R is an “alkyl” group meaning a straightchain, branched or cyclic chemical group containing only carbon andhydrogen. In accordance with an aspect of the present invention, Rincludes without limitation —C₄₋₁₁ H₉₋₂₃, including without limitation—(CH₂)₃₋₁₀CH₃ and straight, branched, cyclic, or any combinationthereof. In accordance with an aspect of the present invention, Rincludes for example methyl, ethyl, propyl, butyl, pentyl, cyclopentyland 2-methylbutyl. R groups are unsubstituted or substituted with 1 ormore substituents (e.g., halogen).

According to one aspect of the present invention, reactive functionalgroups protected by protecting groups are provided directly orindirectly on the surface of a substrate. In one preferred embodiment ofthe present invention, the protecting group is a photoremovable (orphotolabile) protecting group. In another preferred embodiment, theprotecting group is an acid labile protecting group. When a protectinggroup is indirectly on the surface of a substrate, it is generally partof a linker. A linker is a compound that extends from the substratesurface to another compound (e.g., a polymer). Useful linker moleculesare well known to those skilled in the art and representative examplesinclude oligo ethers such as hexaethylene glycol, oligomers ofnucleotides, esters, carbonates, amides and the like.

A “protecting group” is a moiety which may be selectively removed toexpose an active site such as an amino functionality in peptide or aminoacid or a hydroxyl group in a nucleic acid or nucleotide. In accordancewith one aspect of the present invention, protecting groups may beremoved under a variety of condition. For example, an “acid labileprotecting group” is removed by exposure to acid. For an extensivelisting of labile protecting groups useful in the practice of thepresent invention, see also Greene, T. W. and Wuts, P. G. M., ProtectiveGroups in Organic Synthesis, (1991), incorporated herein by reference inits entirety. Useful representative acid sensitive protective groupsinclude dimethoxytrityl (DMT), tert-butylcarbamate (tBoc) andtrifluoroacetyl (tFA). Useful representative base sensitive protectinggroups include 9-fluorenylmethoxycarbonyl (Fmoc), isobutyrl (iBu),benzoyl (Bz) and phenoxyacetyl (pac). Photolabile protecting groupsinclude methyl-6-nitropiperonyloxycarbonyl (MeNPOC), 6-nitroveratryl(NV), 6-nitroveratryloxycarbonyl (NVOC), 6-nitropiperanyl (NP),6-nitropiperonyloxycarbonyl (NPOC), methyl-6-nitroveratryl (MeNV),methyl-6-nitroveratryloxycarbonyl (MeNVOC) and methyl-6-nitropiperonyl(MeNP). Other protecting groups include acetamidomethyl, acetyl,tert-amyloxycarbonyl, benzyl, benzyloxycarbonyl,2-(4-biphenylyl)-2-propyloxycarbonyl, 2-bromobenzyloxycarbonyl,tert-butyl, tert-butyloxycarbonyl,1-carbobenzoxamido-2,2,2-trifluoroethyl, 2,6-dichlorobenzyl,2-(3,5-dimethoxyphenyl)-2-propyloxycarbonyl, 2,4-dinitrophenyl,dithiasuccinyl, formyl, 4-methoxybenzenesulfonyl, 4-methoxybenzyl,4-methylbenzyl, o-nitrophenylsulfenyl, 2-phenyl-2-propyloxycarbonyl,α-2,4,5-tetramethylbenzyloxycarbonyl, p-toluenesulfonyl, xanthenyl,benzyl ester, N-hydroxysuccinimide ester, p-nitrobenzyl ester,p-nitrophenyl ester, phenyl ester, p-nitrocarbonate,p-nitrobenzylcarbonate, trimethylsilyl and pentachlorophenyl ester andthe like.

For acid labile protecting groups, a photoacid generator (“PAG”) isgenerally provided on the surface, preferably in a film with an acidscavenger. See, e.g., U.S. patent application Ser. No. 021700(20050164258), incorporated herein by reference. This is also called a“resist mixture.” The resist mixture can additionally contain asensitizer.

A “photoacid generator” is a compound or substance which produces acid(H⁺ or H₃O⁺) upon exposure to light having a predetermined wavelength.

A “film” as used herein refers to a layer or coating having one or moreconstituents, applied in a generally uniform manner over the entiresurface of a substrate, for example, by spin coating. For example, inaccordance with an aspect of the present invention, a film is asolution, suspension, dispersion, emulsion, or other acceptable form ofa chosen polymer. For example, a film can include a photoacid generatorand optionally a base and a sensitizer, generally in combination with afilm-forming polymer. Film-forming polymers are polymers, which aftermelting or dissolution in a compatible solvent, can form a uniform filmon a substrate.

An “acid scavenger” is a compound or substance which acts to neutralize,adsorb and/or buffer acids, e.g., a base or alkaline compound. Acidscavengers act to reduce the amount or concentration of protons orprotonated water, i.e., H⁺ or H₃O⁺. In the context of the presentinvention, an acid scavenger acts to neutralize, diminish, or bufferacid produced by a photoacid generator. Preferably, an acid scavengerexhibits little or no stratification within a film over time orfollowing exposure to heat. See U.S. Provisional Patent Application No.60/755,261, incorporated herein by reference.

A “sensitizer” is a compound which aids in the use of certain photoacidgenerators (“PAGs”). While the instant invention is not limited by anyparticular mechanism of action or proposed mechanism of action, thesensitizer is understood to extend the photosensitivity of the PAG,i.e., to shift the photo sensitivity to a longer wavelength ofelectromagnetic radiation. The sensitizer, also called aphotosensitizer, is capable of activating the PAG at, for example, alonger wavelength of light in accordance with an aspect of the presentinvention. Preferably, the concentration of the sensitizer is greaterthan that of the PAG, such as 1.1 times to 5 times greater, for example,1.1 times to 3 times greater the concentration of PAG. Exemplarysensitizers suitable for use in the invention includeisopropylthioxanthone (ITX) and 10H-phenoxazine (PhX).

According to an aspect of the present invention, acid is generated inthe selected regions from the PAG by exposure of the PAG to light of apredetermined wavelength. The generated acid contacts the protectedgroup(s) for long enough and under appropriate conditions to remove theprotecting group. In accordance with an aspect of the present invention,the protecting group is preferably a DMT group and it protects ahydroxyl group. The hydroxyl group can be, for example, part of asubstrate, part of a linker, a 5′-hydroxyl group of a nucleotide ordeoxynucleotide or a 3′-hydroxyl group of a nucleotide ordeoxynucleotide. After sufficient exposure of the protective groups tothe acid such that the protective group is removed, but no orsubstantially no damage is done to any polymer, the surface of the arrayis stripped, preferably in an appropriate solvent leaving protected andunprotected groups. In one aspect of the invention, the protectinggroups are exposed to the acid for up to 3 hours, such as up to 1 hour,and typically from 2-30 or 5-15 minutes. In a preferred embodiment ofthe present invention, an acid scavenger is employed in conjunction withthe PAG to limit damage to the polymer, preferably an oligonucleotide,to diminish damage to the polymer from the acid generated from the PAG.

According to one aspect of the present invention, prior to polymerfabrication, the substrate surface is derivatized using a silane mixturein either water or ethanol. The silane applied to the substrate has areactive functional group to serve as a site for the addition ofpolymeric units. Preferably, the silanes also have groups permitting forcross-hybridization of the silanes attached to the substrate, providingadditional stability of the silane layer.

In a further preferred embodiment of the present invention, thecontacting of the surface of the substrate with a silanation compositionis carried out by controlled vapor deposition of the silanationcomposition on a surface of the substrate. Preferably the substrate iscomprised of a glass or silicon substrate. Vapor deposition of thecompositions involves exposure of the substrate to the reagent in avacuum oven. In another aspect of the present invention, the silanationcompositions of the instant invention may also be used in standard bathdeposition or spin coating procedures, as described in greater detailbelow.

In accordance with an aspect of the present invention, silanes areprovided having a protected or “masked” reactive functional group. Inpreferred embodiments of the present invention, the reactive functionalgroups are hydroxyl (—OH) where the polymer is a nucleic acid and amino(—NH2) where the polymer is a peptide or protein. Many silanes withmasked hydroxyl groups are highly volatile. Volatile, masked silanes canbe readily purified by, e.g., distillation, and can be readily employedin gas-phase deposition methods of silanating substrate surfaces. Aftercoating masked silanes onto the surface of the substrate, the maskedreactive functional groups are deprotected to provide an unprotectedreactive functional group.

In accordance with an aspect of the present invention, the masked silanemust have a structure wherein the substrate-silane bond is not cleavedby the demasking reaction or by subsequent steps employed in polymersynthesis.

After demasking, the hydroxyl group of the silane can serve as a sitefor polymer synthesis on the substrate. Preferably the polymer ispeptide or oligonucleotide.

Preferred masked silanes include acetoxyalkylsilanes andepoxyalkylsilanes. A preferred acetoxyalkylsilanes isacetoxyalkyltrichlorosilanes. Particularly preferred masked silanesinclude:

Masked silanes are deprotected in a variety of ways, depending on thestructure of the masking group. Depending on the structure of themasking group, preferred methods of deprotecting include vapor phaseammonia and methylamine or liquid phase aqueous or ethanolic ammonia andalkylamines. In certain cases masked silane reagent may be deprotectedby treatment with aqueous acid.

3-acetoxypropyltrimethoxysilane may be deprotected with nucleophiles,for example ethanolamine and ethylenediamine in ethanol or by hydrolysiswith dilute aqueous acid (aqueous base hydrolysis would deprotect too,but the silane-substrate bonds are generally too subject to cleavageunder basic conditions).

(Trimethoxysilyloxy)-(C₃-C₁₂-alkyl)-(triethoxysilane) may be deprotectedwith dilute aqueous acid solutions.

3-glycidoxypropyltriethoxysilane may be deprotected by ring opening togive a 1,2-diol by acid hydrolysis (aqueous acid).

According to one aspect of the present invention, silanation of thesubstrate comprises dipping or otherwise immersing the substrate in thesilanation composition. Following immersion, the substrate is generallyspun as described for the substrate stripping process, i.e., laterally,to provide a uniform distribution of the composition across the surfaceof the substrate. This ensures a more even distribution of reactivefunctional groups on the surface of the substrate. Following applicationof the silane layer, the silanated substrate may be baked tocross-polymerize the silanes on the surface of the substrate. Bakingtypically takes place at temperatures in the range of from 90° C. to120° C. for a time period of from about 1 minute to about 10 minutes.

In still other preferred embodiments, as noted above, a silanationcomposition is contacted with the surface of the substrate usingcontrolled vapor deposition methods or spray methods. These methodsinvolve the volatilization or atomization of the silanation compositioninto a gas phase or spray, followed by deposition of the gas phase orspray upon the surface of the substrate, usually by ambient exposure ofthe surface of the substrate to the gas phase or spray. Vapor depositionmay result in a more even application of the solution than immersing thesubstrate into the solution.

The efficacy of the silane derivatization process, e.g., the density anduniformity of functional groups on the substrate surface, may generallybe assessed by adding a fluorophore which binds the reactive groups,e.g., a fluorescent phosphoramidite such as Fluoreprime™ from Pharmacia,Corp., Fluoredite™ from Millipore, Corp. or FAM™ from ABI, and lookingat the relative fluorescence across the surface of the substrate.

The stability of a silanated surface can similarly be assessed using thefluorophores described above. In addition, stability can be assessed bymonitoring the hybridization signal from a polymer attached to asilanated surface (the polymer should be selected such that the polymeris stable over the length of the assay).

In accordance with an aspect of the present invention, silanationcompositions used in the invention comprise a mixture of two or moresilanation reagents, wherein at least one silanation reagent includes afunctional group capable of supporting polymer synthesis and at leastone silanation reagent with no functional group capable of supportingpolymer synthesis.

In one embodiment of the present invention, the silanation compositionscomprise a mixture of two or more silanation reagents, wherein at leastone silanation reagent includes a functional group capable of supportingpolymer synthesis and at least one silanation reagent includes nofunctional group capable of supporting polymer synthesis wherein the atleast one silanation reagent including a functional group is notbis(2-hydroxyethyl)-3-aminopropyltriethoxysilane and the at least onesilanation reagent that does not include a functional group is not1,2-bis(trimethoxysilyl)ethane.

According to another aspect of the present invention, the silanationcomposition comprises a mixture of two or more silanation reagents,wherein at least one silanation reagent includes a functional groupcapable of supporting polymer synthesis and at least one silanationreagent which includes no functional group capable of supporting polymersynthesis wherein the silanation reagents are present at predeterminedratios.

According to one aspect of the present invention, it is desired toproduce substrates having varying densities of silanes with functionalgroups. In accordance with an aspect of the present invention arrayshaving various densities of silanes with a functional group is achievedby treating the substrate with mixtures of silane reagent some having afunctional group for polymer synthesis with silanes having no functionalgroup, followed by covalently linking the mixed silanes to thesubstrate.

It has been discovered in accordance with the present invention, it hasbeen discovered that there is a negative correlation between a very highdensity of probes (e.g., oligonucleotides attached to the substrate) andefficiency of target hybridization. Target binding is inhibited by andoverly dense probe concentration on the surface of the substrate by thesteric and electrostatic repulsive effect of overly dense probes on thesurface of the substrate. Inhibition of target binding in turn leads toa decrease in hybridization signal, which can result in a perfect matchbetween target and probe being read by scanner technology as a negativeresult (false negative). It has been discovered in accordance with thepresent invention that one means to control probe density is through thedensity of silanes on the substrate with reactive functional groups. Inaccordance with an aspect of the present invention, the density ofsilanes with functional groups on the surface of the substrate can becontrolled by silanating substrates with silane compositions (mixtures)of silanes with functional groups for polymer synthesis and silaneswithout a functional group for polymer synthesis.

The issue of overly dense probes on the substrate becomes more acute asmethods for in situ fabrication of polymers becomes more efficient. Forexample, photolithographic fabrication of oligonucleotides using theMeNPOC photoprotecting group has less than a 90% efficiency rate ofnucleotide addition in adding additional nucleotides per step infabricating oligonucleotide DNA strands. Due to the recursive nature ofphotolithographic synthesis with photo protecting groups, less than 10%of 25 mer oligonucleotides will achieve full length. This inefficiencyof probe synthesis mitigates inhibition of target/probe binding causedby high density.

However, with the development of higher efficiency photo protectivegroups, some approaching 100% efficiency and the development ofphotoacid generator technology which employs the highly efficient DMTprotective group, high density probes and target/probe hybridizationinhibition is more problematic. To solve this problem, the presentinvention limits the density of silanes having reactive functionalgroups on the substrate by employing silane compositions having silaneswith reactive functional groups and silanes having no functional groupsto limit the density of functional groups on the surface of thesubstrate.

In accordance with an aspect of the present invention, the density ofsilanes with functional groups is controlled by employing silanecompositions with a relatively high molar ratio of non-functional groupsilanes to functional group silanes. In a preferred embodiment of thepresent invention, the molar ratio of the reagent without a functionalgroup to the reagent with a functional group ranges from about 5 to 1 to500 to 1, respectively. More preferably, the ratio is from about 5 to 1to 200 to 1, respectively. Yet another preferred ratio is from about 50to 1 to 100 to 1, respectively. In a particularly preferred aspect ofthe present invention, the ratio is 100 to 1, respectively. In anotherparticularly preferred embodiment of the present invention the ratio is99 to 1.

In a preferred embodiment of the present invention the at least onesilanation reagent without a functional group is1,2-bis(trimethoxysilyl)ethane and the at least one silanation reagentthat includes a functional group isbis(2-hydroxyethyl)-3-aminopropyltriethoxysilane wherein the silanationreagents are present at predetermined ratios. Preferably, the W/V ratioof 1,2-bis(trimethoxysilyl)ethane tobis(2-hydroxyethyl)-3-aminopropyltriethoxysilane group ranges from about5 to 1 to 500 to 1, respectively. More preferably, the ratio is fromabout 5 to 1 to 200 to 1, respectively. Yet another preferred ratio isfrom about 50 to 1 to 100 to 1, respectively. In a particularlypreferred aspect of the present invention, the ratio is 100 to 1,respectively.

In accordance with an aspect of the present invention, functional groupscapable of supporting polymer synthesis generally include nucleophilicor electrophilic functional groups, particularly those such as amino,hydroxyl and carboxylic acid groups. When the polymer is a nucleic acid(e.g., oligonucleotide), the functional group capable of supportingpolymer synthesis is preferably a hydroxyl group. When the polymer is apolypeptide, the functional group capable of supporting polymersynthesis is preferably an amino group. Preferably, a silanation reagentthat lacks functional groups capable of supporting polymer synthesis hasonly alkyl or aryl tertiary amine groups. Preferably, the amines arealkyl tertiary amine groups, aside from the functional groups involvedin silanation of the substrate.

In one embodiment, the at least one silanation reagent which includes afunctional group capable of supporting polymer synthesis is selectedfrom the group consisting of:

wherein R₁ is CH₂ or C(O); R₂ is H or (CH₂)_(n)—Si—(O—(CH₂)_(z)—CH₃)₃; xis 3-7; n is 1 to 5 (e.g., in certain embodiments, n is 1 or 2 when R₂is H); and z is 0-5.

wherein R₃, R₄, and R₅ are independently methyl or ethyl;

wherein R₆, R₇, R₈, R₉, R₁₀, and R₁₁ are methyl or ethyl; and

wherein R₁₂, R₁₃, and R₁₄ are independently methyl or ethyl.

In a preferred embodiment, the at least one silane reagent with afunctional group is selected from the group consisting of:

and the at least one silane reagent without a functional group isselected from the group consisting of alkyl-trimethoxysilane,1,2-bis(trimethoxysilyl)ethane, 1,3-bis(trimethoxysilyl)propane,1,6-bis(trimethoxysilyl)hexane,3-(N,N-dimethylamino)propyltrimethoxysilane andN,N-bis(3-trimethoxysilylpropyl)methylamine.

In a preferred embodiment, the silanation composition includesbis-(3-trimethoxysilylpropyl)methylamine and one ofN-2-hydroxyethyl-N-methyl-3-aminopropyltrimethoxysilane andN-hydroxyethyl-N,N-bis(trimethoxysilylpropyl)amine.

In a preferred embodiment, the silanation composition includesbis(3-trimethoxysilylpropyl)methylamine and one ofN-2-hydroxyethyl-N-methyl-3-aminopropyltrimethoxysilane. The structuresof these compounds are as shown below:

In one embodiment, the silanation reagent are covalently attached to thesurface of a solid substrate to provide a coating comprisingderivatizable functional groups on the substrate, thus permitting arraysof immobilized polymers (preferably oligomers or peptides, morepreferably oligomers) to be covalently attached to the substrate viacovalent reaction with the reactive functional groups. The immobilizedpolymers, such as polypeptides or nucleic acids, can be used in avariety of binding assays including biological binding assays. Inanother embodiment, high density arrays of immobilized nucleic acidprobes may be formed on the substrate, and then one or more targetnucleic acids comprising different target sequences may be screened forbinding to the high density array of nucleic acid probes comprising adiversity of different potentially complementary probe sequences. Forexample, methods for light-directed synthesis of DNA arrays on glasssubstrates is described in McGall et al., J. Am. Chem. Soc.,119:5081-5090 (1997), the disclosure of which is incorporated herein.

Preferably, the array of polymers is an array of nucleic acids. Morepreferably, the array of nucleic acids is an array of oligonucleotides.The monomers for such arrays are preferably naturally or non-naturallyoccurring nucleotides. More preferably, the nucleotides employed in thepresent invention are selected from the group consisting of G, A, T, andC.

According to an aspect of the present invention, a nucleotide isprotected at its 5′ hydroxyl end by a dimethoxytrityl (“DMT”) protectivegroup. In the most preferred embodiments, the nucleotide is selectedfrom the group G, A, T, and C and is protected at its 5′ hydroxyl groupby a DMT protective group. In another aspect of the present invention,the nucleotide is protected at its 3′ hydroxyl group with a DMTprotective group. Thus, in accordance with the present invention,nucleotides may be synthesized in the 5′ to 3′ direction or a 3′ to 5′direction.

According to one aspect of the present invention, linker moleculeshaving reactive functional groups protected by acid labile protectinggroups are provided on the surface of a substrate. In one preferredembodiment of the present invention, a photoacid generator (“PAG”) isprovided on the surface, preferably in a film with an acid scavenger.This is also called a “resist mixture.”

In another aspect of the present invention, the resist mixtureadditionally contains a sensitizer. A set of selected regions on thesurface of the substrate is exposed to radiation using well-knownlithographic methods discussed, for example, in Thompson, L. F.;Willson, C. G.; and Bowden, M. J., Introduction to Microlithography;American Chemical Society, 1994, pp. 212-232, incorporated herein byreference in its entirety.

According to an aspect of the present invention, acid is generated inthe selected regions from the PAG by exposure of the PAG to light of apredetermined wavelength. The generated acid contacts the protectedgroup(s) for long enough and under appropriate conditions to remove theprotective group. In accordance with an aspect of the present invention,the protective group is preferably a DMT group and it protects ahydroxyl group. The hydroxyl group can be, for example, part of asubstrate, part of a linker, a 5′-hydroxyl group of a nucleotide ordeoxynucleotide or a 3′-hydroxyl group of a nucleotide ordeoxynucleotide. After sufficient exposure of the protective groups tothe acid such that the protective group is removed, but no orsubstantially no damage is done to any polymer, the surface of the arrayis stripped, preferably in an appropriate solvent leaving protected andunprotected groups. In one aspect of the invention, the protectivegroups are exposed to the acid for up to 3 hours, such as up to 1 hour,and typically from 2-30 or 5-15 minutes. In a preferred embodiment ofthe present invention, acid exposure is from 1 to 5 minutes.

Monomers having an acid labile protective group are allowed to reactwith the exposed groups from the acid treatment. The surface is againcoated with one of the resist mixtures described above.

In a particular embodiment of the invention, deoxynucleotides having onehydroxyl group with an acid labile protective group and the other with areactive group, preferably a phosphoramidite group, are allowed to reactwith the exposed hydroxyl groups from the acid treatment, allowingcoupling of the nucleotide to the hydroxyl group. The surface is againcoated with one of the resist mixtures described above.

A second set of selected regions is, thereafter, exposed to radiation.The radiation-initiated reactions remove the protecting groups onmolecules in the second set of selected regions, i.e. the linkermolecules and the first-bound monomers. The substrate is then contactedwith a second monomer containing a removable protective group forreaction with exposed functional groups. This process is repeated toselectively apply monomers until polymers of a desired length anddesired chemical sequence are obtained. According to one aspect of thepresent invention, the monomers are preferably nucleotides. Inaccordance with an aspect of the present invention, growing chains ofnucleic acid are preferably capped in between synthesis rounds. Byterminating chain growth where a monomer should have been added but wasnot, capping limits the production of incorrect nucleotide sequences.Side chain protective groups for exocylic amines for example are alsopreferably protected by techniques well known in the art duringsynthesis and deprotected at the conclusion of synthesis of thenucleotide array.

In one preferred embodiment, the monomer is a 2′-deoxynucleosidephosphoramidite containing an acid labile protecting group at its 5′hydroxyl group. Accordingly, a “monomer” is understood to include boththe individual units of a finished polymer (e.g., oligonucleotide,polypeptide) and compounds that become individual units of a finishedpolymer upon attaching to a substrate and optionally further reaction(e.g., to remove protecting groups, to oxidize phosphite esters tophosphate esters). As stated previously, in an alternate embodiment, theprotecting group is present at the 3′ hydroxyl group if synthesis of thepolynucleotide is from the 5′ to 3′ direction. The nucleosidephosphoramidite is represented in accordance with one aspect of thepresent invention by the following formula:

wherein the base is adenine, guanine, thymine, or cytosine, R₁ is aprotecting group which makes the 5′ hydroxyl group unavailable forreaction and includes dimethoxytrityl, tert-butyloxycarbonyl or any ofthe protecting groups known to those of skill in the art; R₂ iscyanoethyl, methyl, t-butyl, trimethylsilyl or the like; and R₃ and R₄are isopropyl, cyclohexyl and the like. Exocyclic amines present on thebases can also be protected with acyl protecting groups such as benzoyl,isobutyryl, phenoxyacetyl and the like. The linker molecule contains anacid- or base-removable protecting group. Useful linker molecules arewell known to those skilled in the art and representative examplesinclude oligo ethers such as hexaethylene glycol, oligomers ofnucleotides, esters, carbonates, amides and the like. Useful protectinggroups include those previously listed and others known to those skilledin the art.

In still another preferred embodiment of the present invention, thearray of polymers is an array of peptides, where the monomers are aminoacids. Suitable amino acids include naturally occurring amino acid andnon-naturally occurring amino acids. Preferably, the amino acid isselected from the group consisting of the L form of alanine, arginine,asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,praline, serine, threonine, tryptophan, tyrosine and valine. Preferably,an amino acid is protected at its amino terminus functionality by atert-butyloxycarbonyl (“tBOC”) protective group during synthesis.

According to another aspect of the present invention, suitable aminoacids include peptide nucleic acids (PNAs). PNAs include a peptidebackbone with nitrogenous bases attached to this backbone, such thatthey can serve as mimics of nucleic acids (including oligomers).Preferably, PNAs have a greater affinity for a complementary nucleicacid sequence than the analogous native nucleic acid. Suitable PNArepeat units are shown by the following structural formulae:

where B represents a base, typically adenine, cytosine, guanine orthymine. Other backbones are suitable, provided that the resulting PNAsare capable of hybridizing with nucleic acids.

Syntheses of PNAs are described in Hyrup and Nielsen, Bioorg. Med. Chem.(1996) 4:5-23; and Vilaivan and Lowe, J. Am. Chem. Soc. (2002)124:9326-9327, the contents of which are incorporated herein byreference.

In an aspect of the invention, density of PNAs in an array and anylinker groups are selected such that a 2:1 complex of PNA to ahybridized DNA or RNA sample can be formed. In another aspect of theinvention, a chimeric polymer of PNA and a nucleic acid is prepared.

In accordance with an aspect of the present invention, a silanationcomposition is provided comprising a mixture of two or more silanationreagents, wherein at least one silanation reagent includes a functionalgroup capable of supporting polymer synthesis and at least onesilanation reagent which includes no functional group capable ofsupporting polymer synthesis.

In preferred embodiment the silanation composition comprises the atleast one silanation reagent which includes a functional group capableof supporting polymer synthesis is selected from the group consistingof:

and the at least one silane reagent which does not include a functionalgroup capable of supporting polymer synthesis is selected from the groupconsisting of alkyl-trimethoxysilane, 1,2-bis(trimethoxysilyl)ethane,1,3-bis(trimethoxysilyl)propane, 1,6-bis(trimethoxysilyl)hexane,3-(N,N-dimethylamino)propyltrimethoxysilane andN,N-bis(3-trimethoxysilylpropyl)methylamine.

In a preferred embodiment, the at least one silanation reagent includinga functional group capable of supporting polymer synthesis is a maskedsilanation reagent. Preferably, the masked silanation reagent isselected from the group consisting of:

In a particularly preferred embodiment the at least one silanationreagent which includes a functional group capable of supporting polymersynthesis is N,N-bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane andthe at least one silanation reagent which includes no functional groupcapable of supporting polymer synthesis is1,2-bis(trimethoxysilyl)ethane.

In a preferred embodiment, the at least one silanation reagent whichincludes a functional group capable of supporting polymer synthesis isN-(2-hydroxyethyl)-N,N-bis(trimethoxysilylpropyl)amine and the at leastone silanation reagent which includes no functional group capable ofsupporting polymer synthesis is 1,2-bis(trimethoxysilyl)ethane.

In a preferred embodiment the at least one silanation reagent whichincludes no functional group capable of supporting polymer synthesis andthe at least one silanation reagent which includes a functional groupcapable of supporting polymer synthesis are present at predeterminedratios. Preferably, the predetermined ratios are weight to volume ratios(W:V). In another preferred embodiment, the ratios are molar ratios.

In a preferred embodiment, the W/V ratio of the at least one silanationreagent which includes no functional group capable of supporting polymersynthesis and the at least one silanation reagent which includes afunctional group capable of supporting polymer synthesis are present atranges from about 5 to 1 to 500 to 1, respectively.

More preferably, the W/V ratio of the at least one silanation reagentwhich includes no functional group capable of supporting polymersynthesis and the at least one silanation reagent which includes afunctional group capable of supporting polymer synthesis are present atranges from about ranges from about 5 to 1 to 200 to 1, respectively.

In another preferred embodiment, the W/V ratio of the at least onesilanation reagent which includes no functional group capable ofsupporting polymer synthesis and the at least one silanation reagentwhich includes a functional group capable of supporting polymersynthesis are present at ranges from about 50 to 1 to 100 to 1,respectively.

More preferably, the W/V ratio of the at least one silanation reagentwhich includes no functional group capable of supporting polymersynthesis and the at least one silanation reagent which includes afunctional group capable of supporting polymer synthesis is about 100 to1 or 99 to 1 respectively.

In a preferred embodiment, the at least one silanation reagent whichincludes no functional group capable of supporting polymer synthesis is1,2-bis(trimethoxysilyl)ethane and the at least one silanation reagentwhich includes a functional group capable of supporting polymersynthesis is bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane.

Preferably, ratio of 1,2-bis(trimethoxysilyl)ethane tobis(2-hydroxyethyl)-3-aminopropyltriethoxysilane ranges from about 5 to1 to 500 to 1, respectively.

More preferably, the W/V ratio of 1,2-bis(trimethoxysilyl)ethane tobis(2-hydroxyethyl)-3-aminopropyltriethoxysilane ranges from about 5 to1 to 200 to 1, respectively.

Still more preferably, the W/V ratio of 1,2-bis(trimethoxysilyl)ethaneto bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane ranges from about 50to 1 to 100 to 1, respectively. Most preferably, the W/V ratio of1,2-bis(trimethoxysilyl)ethane tobis(2-hydroxyethyl)-3-aminopropyltriethoxysilane ratio is about 100 to 1and 99 to 1, respectively.

In another preferred embodiment, the at least one silanation reagentwhich includes no functional group capable of supporting polymersynthesis is bis(3-trimethoxysilylpropyl)methylamine and the at leastone silanation reagent which includes a functional group capable ofsupporting polymer synthesis is selected from the group consisting ofN-2-hydroxyethyl-N-methyl-3-aminopropyltrimethoxysilane andN-2-hydroxyethyl-N,N-bis(trimethoxysilylpropyl)amine.

In another preferred embodiment, the at least one silanation reagentwhich includes no functional group capable of supporting polymersynthesis is bis(3-trimethoxysilylpropyl)methylamine and the at leastone silanation reagent which includes a functional group capable ofsupporting polymer synthesis is selected from the group consisting ofN-2-hydroxyethyl-N-methyl-3-aminopropyltrimethoxysilane andN-2-hydroxyethyl-N,N-bis(trimethoxysilylpropyl)amine and the W:V ratioranges from about 5 to 1 to 500 to 1, respectively.

More preferably, the W:V ratio ofbis(3-trimethoxysilylpropyl)methylamine toN-2-hydroxyethyl-N-methyl-3-aminopropyltrimethoxysilane orN-2-hydroxyethyl-N,N-bis(trimethoxysilylpropyl)amine ranges from about 5to 1 to 200 to 1, respectively.

Still more preferably, the W:V ratio ofbis(3-trimethoxysilylpropyl)methylamine toN-2-hydroxyethyl-N-methyl-3-aminopropyltrimethoxysilane orN-2-hydroxyethyl-N,N-bis(trimethoxysilylpropyl)amine ranges from about50 to 1 to 100 to 1, respectively.

Most preferably, the W:V ratio ofbis(3-trimethoxysilylpropyl)methylamine toN-2-hydroxyethyl-N-methyl-3-aminopropyltrimethoxysilane orN-2-hydroxyethyl-N,N-bis(trimethoxysilylpropyl)amine is about 100 to 1and 99 to 1, respectively.

In another preferred embodiment, the composition does not comprise amixture of bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane and1,2-bis(trimethoxysilyl)ethane. In another preferred embodiment thecomposition (e.g., of claim 1) does not contain1,2-bis(trimethoxysilyl)ethane.

In accordance with an aspect of the present invention, a method ofsilanating a surface is presented wherein a silica or other siliconoxide-containing surface with a silanation composition that includes amixture of two or more silanation reagents, where at least onesilanation reagent which includes no functional group capable ofsupporting polymer synthesis and at least one silanation reagentincludes a functional group capable of supporting polymer synthesis.

A preferred embodiment of the method of silanating a surface, whereinthe at least one silanation reagent which includes a functional groupcapable of supporting polymer synthesis is selected from the groupconsisting of:

and wherein the at least one silanation reagent which includes nofunctional group capable of supporting polymer synthesis is selectedfrom the group consisting of alkyltrimethoxysilane,1,2-bis(trimethoxysilyl)ethane, 1,3-bis(trimethoxysilyl)propane,1,6-bis(trimethoxysilyl)hexane,3-(N,N-dimethylamino)propyltrimethoxysilane andN,N-bis(3-trimethoxysilylpropyl)methylamine.

In a preferred embodiment of the invention, the at least one silanationreagent which includes a functional group capable of supporting polymersynthesis is N-(2-hydroxyethyl)-N,N-bis(trimethoxysilylpropyl)amine andthe at least one silanation reagent which includes no functional groupcapable of supporting polymer synthesis is1,2-bis(trimethoxysilyl)ethane.

In a preferred embodiment, the method of silanating a surface employs asthe at least one silanation reagent which includes a functional groupcapable of supporting polymer synthesis comprises a masked silanationreagent.

Preferably, the masked silanation reagent is selected from the groupconsisting of:

In another preferred embodiment, the method of silanating a surface isby vapor deposition of the masked silanating reagent.

Preferably, the method of silanating a surface with a masked silanatingreagent further comprises the step of deprotecting the masked silanatingreagent to provide a deprotected functional group capable of supportingpolymer synthesis.

In a preferred embodiment of the method of silanating a surface the atleast one silanation reagent which includes no functional group capableof supporting polymer synthesis isbis(3-trimethoxysilylpropyl)methylamine and the at least one silanationreagent which includes a functional group capable of supporting polymersynthesis is selected from the group consisting ofN-2-hydroxyethyl-N-methyl-3-aminopropyltrimethoxysilane andN-hydroxyethyl-N,N-bis(trimethoxysilylpropyl)amine.

In another preferred embodiment, in the method of silanating a surfacethe silanating composition does not comprise a mixture ofbis(2-hydroxyethyl)-3-aminopropyltriethoxysilane and1,2-bis(trimethoxysilyl)ethane. In another preferred embodiment, thecomposition does not contain 1,2-bis(trimethoxysilyl)ethane.

In accordance with another aspect of the present invention, a method ispresented of preparing an array of polymers comprising: providing asubstrate; contacting a silica or other silicon oxide-containing surfaceof the substrate with a silanation composition that comprises a mixtureof two or more silanation reagents, where at least one silanationreagent includes a functional group capable of supporting polymersynthesis and at least one silanation reagent includes no functionalgroup capable of supporting polymer synthesis; protecting the functionalgroup capable of supporting polymer synthesis with a protecting group orreacting the functional group capable of supporting polymer synthesiswith a compound having a reactive group protected by a protecting group;removing the protecting group in selected regions of the surface toprovide an exposed functional or reactive group; reacting the exposedgroup with a monomer, wherein the monomer is coupled to the exposedgroup; and repeating the steps of removing and reacting to produce thearray of polymers.

Preferably, the array of polymers is an array of nucleic acids. It isalso preferred that the array of polymers is a polypeptide array.

In a preferred embodiment of the method of preparing an array ofpolymers, the silanation composition comprisesbis(3-trimethoxysilylpropyl)methylamine and one ofN-2-hydroxyethyl-N-methyl-3-aminopropyltrimethoxysilane andN-2-hydroxyethyl-N,N-bis(trimethoxysilylpropyl)amine.

In another preferred embodiment of the method of preparing an array ofpolymers, the silanation composition comprisesbis(3-trimethoxysilylpropyl)methylamine andN-2-hydroxyethyl-N-methyl-3-aminopropyltrimethoxysilane.

In another preferred embodiment of the method of preparing an array ofpolymers, the silanation composition comprisesbis(3-trimethoxysilylpropyl)methylamine andN-2-hydroxyethyl-N,N-bis(trimethoxysilylpropyl)amine.

In a preferred embodiment of the method of preparing an array ofpolymers, the protecting group is a photoremovable protecting group.

In still another preferred embodiment of the method of preparing anarray of polymers, the protecting group is an acid labile protectinggroup.

In still another preferred embodiment of the method of preparing anarray of polymers, the step of removing comprises activating a photoacidgenerator in the selected regions by selective application of lighthaving a predetermined wavelength to provide an acid and exposing theprotecting group to the acid.

In still another preferred embodiment of the method of preparing anarray of polymers, the at least one silanation reagent which includes afunctional group capable of supporting polymer synthesis is selectedfrom the group consisting of:

and wherein the at least one silane reagent which does not include afunctional group capable of supporting polymer synthesis is selectedfrom the group consisting of alkyltrimethoxysilane,1,2-bis(trimethoxysilyl)ethane, 1,3-bis(trimethoxysilyl)propane,1,6-bis(trimethoxysilyl)hexane,3-(N,N-dimethylamino)propyltrimethoxysilane andN,N-bis(3-trimethoxysilylpropyl)methylamine.

In still another preferred embodiment of the method of preparing anarray of polymers, the at least one silanation reagent which includes afunctional group capable of supporting polymer synthesis comprises amasked silanation reagent.

Preferred masked silanation reagents include:

Preferably, masked silanating reagents are applied by vapor depositionof the masked silanating reagent. The masked silanating reagents arepreferably deprotected to provide a deprotected functional group capableof supporting polymer synthesis.

In still another preferred embodiment of the method of preparing anarray of polymers, the silanation composition comprises a mixture of twoor more silanation reagents, wherein the at least one silanation reagentwhich includes a functional group capable of supporting polymersynthesis is N,N-bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane andthe at least one silanation reagent which includes no functional groupcapable of supporting polymer synthesis is1,2-bis(trimethoxysilyl)ethane.

In still another preferred embodiment of the method of preparing anarray of polymers, the at least one silanation reagent which includes afunctional group capable of supporting polymer synthesis isN-(2-hydroxyethyl)-N,N-bis(trimethoxysilylpropyl)amine and the at leastone silanation reagent which includes no functional group capable ofsupporting polymer synthesis is 1,2-bis(trimethoxysilyl)ethane.

In still another preferred embodiment of the method of preparing anarray of polymers, the at least one silanation reagent which includes nofunctional group capable of supporting polymer synthesis and at theleast one silanation reagent which includes a functional group capableof supporting polymer synthesis are present at predetermined ratios.

Preferably, the predetermined ratios are molar ratios.

In still another preferred embodiment of the method of preparing anarray of polymers, the W/V ratio of the at least one silanation reagentwhich includes no functional group capable of supporting polymersynthesis and the at least one silanation reagent which includes afunctional group capable of supporting polymer synthesis are present atranges from about 5 to 1 to 500 to 1, respectively.

More preferably, the W/V ratio ranges from about 5 to 1 to 200 to 1,respectively. Still more preferably, the ratio ranges from about 50 to 1to 100 to 1, respectively. Most preferably, the ratio is about 100 to 1and 99 to 1, respectively.

In still another preferred embodiment of the method of preparing anarray of polymers, the at least one silanation reagent which includes nofunctional group capable of supporting polymer synthesis is1,2-bis(trimethoxysilyl)ethane and the at least one silanation reagentwhich includes a functional group capable of supporting polymersynthesis is bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane.

In still another preferred embodiment of the method of preparing anarray of polymers, the W/V ratio of 1,2-bis(trimethoxysilyl)ethane tobis(2-hydroxyethyl)-3-aminopropyltriethoxysilane ranges from about 5 to1 to 500 to 1, respectively. More preferably, the W/V ratio of1,2-bis(trimethoxysilyl)ethane tobis(2-hydroxyethyl)-3-aminopropyltriethoxysilane ranges from about 5 to1 to 200 to 1, respectively. Still more preferably, the W/V ratio of1,2-bis(trimethoxysilyl)ethane tobis(2-hydroxyethyl)-3-aminopropyltriethoxysilane ranges from about 50 to1 to 100 to 1, respectively. Most preferably, the W/V ratio of1,2-bis(trimethoxysilyl)ethane tobis(2-hydroxyethyl)-3-aminopropyltriethoxysilane ratio is about 100 to1, respectively.

In an other preferred embodiment of the method of preparing an array ofpolymers, the silanating composition does not comprise a mixture ofbis(2-hydroxyethyl)-3-aminopropyltriethoxysilane and1,2-bis(trimethoxysilyl)ethane. In another preferred embodiment, thecomposition does not contain 1,2-bis(trimethoxysilyl)ethane.

According to an aspect of the present invention, an array of polymers ona surface of a substrate is presented, where the surface of thesubstrate is silanated by a silanation composition that includes amixture of two or more silanation reagents, where at least onesilanation reagent includes a functional group capable of supportingpolymer synthesis and at least one silanation reagent includes nofunctional group capable of supporting polymer synthesis.

Preferably the polymer is a nucleic acid. The polymer is also preferablya polypeptide.

In a preferred embodiment of the array of polymers on a surface of asubstrate, the silanation composition comprisesbis(3-trimethoxysilylpropyl)methylamine and one ofN-2-hydroxyethyl-N-methyl-3-aminopropyltrimethoxysilane andN-2-hydroxyethyl-N,N-bis(trimethoxysilylpropyl)amine.

In a preferred embodiment of the array of polymers on a surface of asubstrate, the at least one silanation reagent which includes afunctional group capable of supporting polymer synthesis is selectedfrom the group consisting of:

and wherein the at least one silane reagent which does not include afunctional group capable of supporting polymer synthesis is selectedfrom the group consisting of alkyltrimethoxysilane,1,2-bis(trimethoxysilyl)ethane, 1,3-bis(trimethoxysilyl)propane,1,6-bis(trimethoxysilyl)hexane,3-(N,N-dimethylamino)propyltrimethoxysilane andN,N-bis(3-trimethoxysilylpropyl)methylamine.

In a preferred embodiment of the array of polymers on a surface of asubstrate, the at least one silanation reagent which includes afunctional group capable of supporting polymer synthesis comprises amasked silanation reagent.

Preferred masked silanation reagents include:

Preferably, the masked silanation reagent is deprotected to provide adeprotected functional group capable of supporting polymer synthesis.

In a preferred embodiment of the array of polymers on the surface of asubstrate, the at least one silanation reagent which includes afunctional group capable of supporting polymer synthesis isN,N-bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane and the at leastone silanation reagent which includes no functional group capable ofsupporting polymer synthesis is 1,2-bis(trimethoxysilyl)ethane.

In a preferred embodiment of the array of polymers on the surface of asubstrate, the at least one silanation reagent which includes afunctional group capable of supporting polymer synthesis isN-(2-hydroxyethyl)-N,N-bis(trimethoxysilylpropyl)amine and the at leastone silanation reagent which includes no functional group capable ofsupporting polymer synthesis is 1,2-bis(trimethoxysilyl)ethane.

In a preferred embodiment of the array of polymers on the surface of asubstrate, the at least one silanation reagent which includes nofunctional group capable of supporting polymer synthesis and at theleast one silanation reagent which includes a functional group capableof supporting polymer synthesis are present at predetermined ratios.

Preferably, the predetermined ratios are molar ratios. In a preferredembodiment of the array of polymers on the surface of a substrate, themolar ratio of the at least one silanation reagent which includes nofunctional group capable of supporting polymer synthesis and the atleast one silanation reagent which includes a functional group capableof supporting polymer synthesis are present at ranges from about 5 to 1to 500 to 1, respectively. More preferably, the W/V ratio ranges fromabout 5 to 1 to 200 to 1, respectively. Still more preferably, the ratioranges from about 50 to 1 to 100 to 1, respectively. Most preferably,the ratio is about 100 to 1 and 99 to 1, respectively.

In a preferred embodiment of the array of polymers on the surface of asubstrate, the at least one silanation reagent which includes nofunctional group capable of supporting polymer synthesis is1,2-bis(trimethoxysilyl)ethane and the at least one silanation reagentwhich includes a functional group capable of supporting polymersynthesis is bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane.

In a preferred embodiment of the array of polymers on the surface of asubstrate, the W/V ratio of 1,2-bis(trimethoxysilyl)ethane tobis(2-hydroxyethyl)-3-aminopropyltriethoxysilane ranges from about 5 to1 to 500 to 1, respectively. Still more preferably, the W/V ratio of1,2-bis(trimethoxysilyl)ethane tobis(2-hydroxyethyl)-3-aminopropyltriethoxysilane ranges from about 5 to1 to 200 to 1, respectively. More preferably, the W/V ratio of1,2-bis(trimethoxysilyl)ethane tobis(2-hydroxyethyl)-3-aminopropyltriethoxysilane ranges from about 50 to1 to 100 to 1, respectively. Most preferably, the W/V ratio of1,2-bis(trimethoxysilyl)ethane tobis(2-hydroxyethyl)-3-aminopropyltriethoxysilane ratio is about 100 to1, respectively.

In another preferred embodiment of the array of polymers, the silanatingcomposition does not comprise a mixture ofbis(2-hydroxyethyl)-3-aminopropyltriethoxysilane and1,2-bis(trimethoxysilyl)ethane. In another preferred embodiment, thecomposition does not contain 1,2-bis(trimethoxysilyl)ethane.

According to an aspect of the present invention, a method is presentedof silanating a substrate with mixture of two or more silanationreagents, where at least one silanation reagent which includes nofunctional group capable of supporting polymer synthesis and at leastone silanation reagent includes a functional group capable of supportingpolymer synthesis, the substrate comprising a silica or other siliconoxide-containing surface, the steps of the method include

a. providing a substrate;

b. immersing the substrate into anhydrous ethanol for a set period oftime;

c. transferring the substrate into a container of silane mixture, thesilane mixture comprising at least one silanation reagent which includesno functional group capable of supporting polymer synthesis and at leastone silanation reagent includes a functional group capable of supportingpolymer synthesis for a set time; and

d. rinsing the substrate with 2-propanol.

Preferably, the silane mixture is 1:99 mole % Bisb silane and BTSMEsilane. In a preferred embodiment the silane mixture of Bisb silane andBTSME silane and further contains anhydrous alcohol, deionized water,and the set time is about 60 minutes.

EXAMPLES I. Substrate Silanation Using a 1:99 Molar Bisb:BTMSE Mixture

The following procedure was used to silanate wafers for R&D andexperimental purposes.

Definitions:

-   -   Bisb: N-(2-hydroxyethyl)-N,N-bis(trimethoxysilylpropyl)amine in        65% methanol.    -   BTMSE: Bis(trimethoxysilyl)ethane    -   SRD: Spin Rinse Dryer    -   DI H₂O: Deionized Water    -   RPM: Revolutions per Minute    -   Substrates: Wafers or slides        Procedure:

Four 8×8″ tanks that have been cleaned and dried were provided. Thetanks were designated as follows: Tank #1 Reagent Alcohol, Tank #2Silane Bath, Tank #3 2-Propanol Bath Rinse #1 and Tank #4 2-PropanolBath Rinse #2

Tank #1 was prepared by adding 6.0 L of Reagent Alcohol to the tank.

Tank #2 (1:99 mol % Bisb:BTMSE Silane Solution) was prepared as follows:5610 mL of reagent alcohol was mixed with 310 mL of DI H₂O and stirredfor 3 minutes. After stirring, 40 mL of BTMSE crosslinker into a 100 mLgraduated cylinder and slowly added to Tank #2. 40 mL of reagent alcoholwas slowly poured into Tank #2 and stirred for 3 minutes. Finally, 780uL of Bisb Silane was slowly added to Tank #2 and followed by stirringthe mixture for a minimum of 4 hours-maximum 12 hours before Silanation.

Tanks #3 and 4 were both employed to rinse the substrate with2-propanol, each consisted of 6 L of the alcohol.

Silanation was conducted by first transferring the substrates to besilanated into weighted substrate cassettes. The cassettes were immersedseveral times into Tank #1, “Reagent Alcohol” for a total of about 3minutes.

After the 3-minute soak in Tank #1, the cassettes were immediatelytransferred to Tank #2, “Silane”, so that the entire substrate wassubmerged. An orbital shaker was employed and the cassettes were leftexposed to Tank #2 for about 60 minutes.

Immediately after expiration of the 60 minutes, the cassettes weretransferred to to Tank #3, “2-Propanol” rinse #1 for 5 minutes. Thecassettes were then transferred to Tank #4, “2-Propanol” rinse for 5minutes.

II. Functional and Non-Functional Silanes

Various functional and non-functional silanes were prepared at differentmolar ratios to test which mixtures provided an appropriate hydroxyldensity (pmol/cm²). Two functional and two non functional silanes aredepicted in FIG. 1.

III. Formulations for Silanation R2+Bisb

To test which molar ratios of non-functional silanes to functionalsilanes provided the desired hydroxyl density, various molar ratios ofsilanation solution were prepared. These are depicted in FIG. 2.

IV. Functional Hydroxyl Density Determination R2/Bisb

Functional hydroxyl density on the substrate was determined using thesilane mixtures of Example 2. The results are depicted in FIG. 3.

V. Molar Ratio of Functional and Non-Functional Silanes

To determine appropriate molar ratios of two other functional andnon-functional silanes, three different silanation solutions with threedifferent molar ratios of R2/R1 (98, 50, and 10) were prepared. Thecompositions are depicted in FIG. 4.

VI. Functional Hydroxyl Density Determination R2/R1

The hydroxyl density in pmol/cm² on the substrate provided by the R2/R1solutions was determined and the data presented in FIG. 5.

VII. Fabrication of High Density Arrays with Functional andNon-Functional Silanes

To determine whether silanation with non-functional and functionalsilanes using a high proportion of nonfunctional to functional silanesprovided improved hybridization results high density arrays wereconstructed. One high density array having 3 micron features and 25 merprobes was fabricated using two silane solutions, including Bisb (afunctional silane) at approximately 60 pmol/cm² on the substrate.

A second array was constructed using a 1:99 Bisb:BTMSE (a non-functionalsilane), which provided a functional hydroxyl density of approximately20 pmol/cm2. The second array also had 3 micron features and 25 merprobes.

Both arrays were hybridized to 20 mM DNA target/SAPE for 16 hours. TheBisb array after 16 hours of hybridization gave no signal and abackground intensity of 85. In contrast, the Bisb:BTMSE (1:99 ratio)gave high signal hybridization results (0, 60,000) display range andgood signal (intensity approximately 60,000).

To determine if the Bisb array could yield better signal with longerhybridization, a Bisb array, identical to that described above washybridized to 20 mM DNA/SAPE for 112 hours. Signal did improve after the112 hour hybridization, but to only 0, 20,000 display range, one thirdof the Bisb:BTMSE array hybridized for only 16 hours. Intensity improvedas well, but only to 16,000 as opposed to 40,000 for the Bisb:BTMSE.

The Bisb:BTMSE (1:99 ratio) silane composition thus provideddramatically increased signal as compared to the Bisb array. Whilehybridization results could be improved for the Bisb array byhybridizing for 112 hours, the display range and signal intensity werestill substantially below the Bisb:BTMSE array after only 16 hourshybridization. In addition, a 112 hour hybridization is impractical forregular experimentation with high density arrays.

The results of these experiments are depicted in FIG. 6.

All publications, patents and patent applications referred to herein areincorporated herein by reference in their entirety.

The above description is illustrative and not restrictive. Manyvariations of the invention will become apparent to those of skill inthe art upon review of this disclosure. Merely by way of example avariety of substrates, polymers, initiators, synthesis initiation sites,and other materials may be used without departing from the scope of theinvention.

The invention claimed is:
 1. An array of polymers on a silanated glass or polymer substrate including a silica surface thereon, wherein the silica surface has covalently attached thereto a silanation coating comprising a mixture of a first compound and a second compound, and wherein the first and second compounds are each covalently attached to the silica surface; the first compound comprising a trialkoxy silyl group and wherein the first compound further includes a reactive functional group capable of supporting polymer synthesis selected from the group consisting of hydroxyl, amino and carboxyl, and wherein the polymers of the array are attached to the silanation coating via the active functional group of the first compound; the first compound being selected from the group consisting of N-(3-(triethoxysilyl)-propyl)-4-hydroxybutyramide; N,N-bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane; 3-((2-hydroxyethyl)methylamino)propyl-trimethoxysilane, N-(2-hydroxy-ethyl)-N,N-bis(trimethoxy-silylpropyl)-amine; 1,2-bis((2-hydroxyethyl)-(3-trimethoxysilylpropyl)aminoethane; 3-acetoxy-propyltrimethoxysilane; (trimethoxysilyloxy)-(C3-C12-alkyl)-(triethoxysilane); and 3-glycidoxypropyltriethoxysilane; and the second compound comprising a trialkoxy silyl group and wherein the second compound does not include a reactive functional group of the first compound; the second compound being selected from the group consisting of 1,2-bis(trimethoxysilyl)-ethane; 1,3-bis(trimethoxysilyl)propane; 1,6-bis(trimethoxysilyl)hexane; 3-(N,N-dimethyl-amino)-propyl-trimethoxysilane; and N,N-bis(3-trimethoxysilylpropyl)-methylamine; wherein the molar ratio of the first compound to the second compound ranges from about 1 to 50 to 1 to 500; and wherein the mixture of the first and second compounds in the silanation coating imparts increased hydrolytic stability to the coating.
 2. The array of polymers according to claim 1, wherein the first compound is N-(3-(triethoxysilyl)propyl)-4-hydroxybutyramide.
 3. The array of polymers according to claim 1, wherein the first compound is N,N-bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane.
 4. The array of polymers according to claim 1, wherein the first compound is 3-((2-hydroxyethyl)methylamino)propyltrimethoxysilane.
 5. The array of polymers according to claim 1, wherein the first compound is N-(2-hydroxyethyl)-N,N-bis(trimethoxysilylpropyl)amine.
 6. The array of polymers according to claim 1, wherein the first compound is 1,2-bis((2-hydroxyethyl)(3-trimethoxysilylpropyl)aminoethane.
 7. The array of polymers according to claim 1, wherein the first compound is 3-acetoxypropyltrimethoxysilane.
 8. The array of polymers according to claim 1, wherein the first compound is (trimethoxysilyloxy)-(C3-C12-alkyl)-(triethoxysilane).
 9. The array of polymers according to claim 1, wherein the first compound is 3-glycidoxypropyltriethoxysilane.
 10. The array of polymers according to claim 1, wherein the second compound is 1,2-bis(trimethoxysilyl)ethane.
 11. The array of polymers according to claim 1, wherein the second compound is bis(trimethoxysilyl)propane.
 12. The array of polymers according to claim 1, wherein the second compound is 1,6-bis(trimethoxysilyl) hexane.
 13. The array of polymers according to claim 1, wherein the second compound is 3-(N,N-dimethylamino)propyltrimethoxysilane.
 14. The array of polymers according to claim 1, wherein the second compound is N,N-bis(3-trimethoxysilylpropyl)-methylamine.
 15. The array of polymers according to claim 1, wherein the polymers are nucleic acids.
 16. The array of polymers according to claim 1, wherein the functional group of the first compound capable of supporting polymer synthesis is protected with a protecting group before the polymers of the array are attached to the silanated substrate.
 17. The array of polymers according to claim 16, wherein the protecting group is an acid labile protecting group.
 18. The array of polymers according to claim 17, wherein the protecting group is removed before the polymers of the array are attached to the silanated substrate by activation of a photoacid generator in selected regions of the silica surface by selective application of light having a predetermined wavelength to provide an acid and exposure of the protecting group to the acid.
 19. The array of polymers according to claim 1, wherein the first compound is N-(2-hydroxyethyl)-N,N-bis(trimethoxysilylpropyl)amine and the second compound is 1,2-bis(trimethoxysilyl)ethane.
 20. The array of polymers according to claim 1, wherein the ratio of the second compound to the first compound ranges from about 50 to 1 to 100 to
 1. 21. The array of polymers according to claim 20, wherein the ratio of the second compound to the first compound is about 99 to
 1. 22. The array of polymers according to claim 1, wherein the second compound is 1,2-bis(trimethoxysilyl)ethane and the first compound is bis(2-hydroxyethyl)-3-aminopropyltriethoxy silane.
 23. The array of polymers according to claim 22, wherein the molar ratio of 1,2-bis(trimethoxysilyl)ethane to bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane ranges from about 50 to 1 to 100 to
 1. 24. The array of polymers according to claim 23, wherein the molar ratio of 1,2-bis(trimethoxysilyl)ethane to bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane ratio is about 99 to
 1. 25. The array of polymers according to claim 1, wherein the silanation coating does not comprise a mixture of bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane and 1,2-bis(trimethoxysilyl)ethane.
 26. The array of polymers according to claim 1, wherein the silanation coating does not contain 1,2-bis(trimethoxysilyl)ethane.
 27. The array of polymers of claim 1, further comprising a hexaethylene glycol linker extending from the functional group capable of supporting polymer synthesis to the polymers of the array. 